Method for diagnosis and monitoring of viral infection by analysis of viral transrenal nucleic acids in urine

ABSTRACT

The present invention relates to methods for diagnosis or monitoring of viral infection by detecting the presence of transrenal viral nucleic acids or nucleic acids of viral origin in urine sample, with or without isolation of nucleic acids from a urine sample. The analysis of the nucleic acids is performed through hybridization of the nucleic acids with specific probes, or through a chain amplification reaction with specific primers. The methods are applicable to all viral pathogenic agents, including RNA, DNA, episomal, or integrated viruses.

RELATED APPLICATIONS

This application claims priority to Italian patent applicationRM2005000067, filed Feb. 17, 2005, bearing attorney docket number6626PTIT, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The technical field of the invention is that of the molecular diagnosisof viral infections.

BACKGROUND OF THE INVENTION

Early analysis and the possibility of directly genotyping the pathogenicagent are among the principal objectives of research in the diagnosticfield. The development of new diagnostic assays should also take intoaccount a degree of compatibility to high-throughput screening methodsand a high level of sensitivity for diagnoses made as soon as possibleafter the occurrence of the infection. In developing countries it isalso important to take into account the ease of handling of biologicalsamples, for more widespread distribution of the diagnostic assays.

There are currently three types of in vitro diagnostic systems: directculture of the pathogenic agent from the biological sample, which is theso-called “gold standard” of diagnostic assays; immunological assaysbased on the detection of products or antigens of the infectious agent;and indirect immunological assays that can detect antibodies producedagainst the infectious agent during or after infection.

In the first system, the principal disadvantage is that the biologicalsample must be considered to be at risk, inasmuch as it can potentiallytransmit the pathogenic agent, whereas in the indirect detection of theantibodies there is no possibility of discriminating between past andcurrent infections.

Molecular diagnostic methods have been developed that are based on thedetection of the nucleic acids of the pathogenic agent in the blood orplasma samples, or in the cell cultures, taken from the patient. Thesetests are usually much more sensitive than the immunoassays. For thisreason, and because of their specificity, they are extremely promising,but usually require special equipment and qualified personnel.

Molecular diagnostic methods based on transrenal DNA (TrDNA) have beendescribed and their utility for monitoring the fate of allogeneictransplants, to detect the sex of a fetus, and to screen the presence oftumor markers was demonstrated. In particular, U.S. Pat. No. 6,251,638describes an analytical method for detecting male fetal DNA in the urineof pregnant women; in U.S. Pat. No. 6,287,820, the invention is aimed atthe diagnosis of tumors, particularly of adenocarcinomas (of the colonand pancreas); and in U.S. Pat. No. 6,492,144, the transrenalnucleic-acid analysis method is used to monitor the progress ofallogeneic transplants, using known methods for molecular analysis. Thepresence of identifiable transrenal DNA in urine, in the fraction of DNAfragments consisting of 150 base pairs or more, was shown (Al-Yatama etal. (2001), “Detection of Y-chromosome-specific DNA in the plasma andurine of pregnant women using nested polymerase chain reaction”; PrenatDiagn, 21:399-402; and Utting, M., et al. (2002), “Microsatelliteanalysis of free tumor DNA in urine, serum, and plasma of patients: Aminimally invasive method for the detection of bladder cancer”; ClinCancer Res, 8:35-40).

Molecular detection of TrDNA in urine is performed using techniques thatare very well known in the art and widely used in laboratory practice,such as PCR (polymerase chain reaction), hybridization, or the so-called“cycling probe reaction.”

The presence of transrenal DNA has been explained as being the result ofphenomenon of apoptosis. In the process of apoptosis or programmed celldeath the nuclear DNA is cleaved into nucleosomes and oligomers, whichsubsequently, as a part of apoptotic process, are phagocytozed andremoved from the organism. (Umansky, S. R., et al. (1982), “In vivo DNAdegradation in thymocytes of gamma-irradiated or hydrocortisone-treatedrats”; Biochim. Biophys. Acta, 655:9-17). A portion of this degradedDNA, though, escapes the phagocytosis, and appears in the bloodstream(Lichtenstein, A. V., et al. (2001), “Circulating nucleic acids andapoptosis”; Ann NY Acad Sci, 945:239-249), and, as confirmed in theabove-referred patents, also in urine.

The presence of viral DNA that originates from sources outside of theurinary tract, in urine has not been described until now. Meanwhile,circulation of viral DNA released from the genome of transfected cell inthe plasma has been shown: for example, fragments of Epstein-Barr viralDNA were detected in plasma of patients with nasopharyngeal carcinoma(Chan, K. C., et al. (2002), “Molecular characterization of circulatingEBV DNA in the plasma of nasopharyngeal carcinoma and lymphomapatients”; Cancer Res 63:2028-2032), and in the case of human papillomavirus (HPV) in the plasma of patients with cervical cancer(Pornthanakasem, W., et al. (2001), “Human Papillomavirus DNA in plasmaof patients with cervical cancer”; BMC Cancer 1:2).

SUMMARY OF THE INVENTION

The present invention relates to methods for diagnosis and monitoring ofviral infections by detecting and quantifying transrenal viralNA-specific sequences. In embodiments, the nucleic acids are isolated orpurified. The method also includes the fractionation of the urinesample, for example, through centrifugation or filtration, with theseparation of a cell-free fraction from a fraction associated with thecell bodies.

Furthermore, in another embodiment, the sample is pretreated with adenaturing agent.

The analysis of the nucleic acids is performed using one of thefollowing techniques: hybridization of the nucleic acids, the cyclingprobe reaction, a polymerase chain reaction, a nested polymerase chainreaction, single-strand conformation polymorphism, a ligase chainreaction, strand displacement amplification, and restriction fragmentlength polymorphism.

The method is applicable to all viral pathogenic agents, including RNA,DNA, episomal, and integrative viruses. It also applies to recombinantviruses, such as the adenoviruses or lentiviruses utilized in genetherapy. In particular, the methods apply to the following viruses:retroviruses, including recombinant and natural HIV-1, HIV-2, variolavirus, poliovirus, herpes simplex virus (HSV), Epstein-Barr virus (EBV),hepatitis C virus (HCV), hepatitis B virus (HBV) and adenoviruses (AAV).In some embodiments the viral agents are Epstein-Barr virus (EBV) andHIV-1.

In another of its embodiments, the invention relates to a kit for thedetection of viral nucleic acid in urine, including: reagents and/ormaterials for the fractionation and/or extraction of transrenal nucleicacids from urine, DNA probe, or pairs of specific oligonucleotides(primers) for at least one viral agent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention relates. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photographic image showing the results of gelelectrophoresis of the amplification product obtained through nested PCRof the transrenal DNA of non-fractionated urine samples of patientsinfected with HIV-1 (20 cycles with the 468/602 primer pair [134 bp]followed by 35 cycles with 518/596 [79 bp]). The primers are specificfor the HIV-1 GAG region.

FIG. 2 is a photographic image showing the results of gelelectrophoresis of the amplification product obtained through nested PCRof the genomic DNA extracted from 8E5 LAV cells (20 cycles with the468/602 primer pair [134 bp] followed by 35 cycles with 518/596 [79bp]). The primers are specific for the HIV-1 GAG region.

FIG. 3 is a photographic image showing the results of gelelectrophoresis of the amplification product obtained through nested PCRof the transrenal DNA of urine samples of patients infected with HIV-1(CP: complete; SN: supernatant; PT: pellet). A-102 bp/60 bp (specificprimers for the HIV-1 POL region); B-317 bp/60 bp (specific for POL);C-569 bp/132 bp (specific for TAT); NI: non-infected; IN 1-3: patientsinfected with HIV.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present definitions are offered for the purposes of the presentinvention:

Amplicon: A term for any relatively small, DNA fragment that isreplicated, e.g., by PCR.

Amplification: An increase in the number of copies of a specific DNAfragment can occur in vivo or in vitro.

Apoptosis: Programmed cell death in normally functioning human andanimal cells when age or state of cell health and condition dictate. Anactive process requiring metabolic activity by the dying cell,characterized by cleavage of the DNA into fragments that give a socalled laddering pattern of DNA fragments of nucleosomal size and itsoligomers.

Chaotropic: The property of chemical substances (e.g., ions such asSCN⁻, ClO₄ ⁻, and guanidine) that disturb the thermodynamic structure ofwater. It allows less polar and more hydrophobic substances to becomemore soluble in water. The effect at the biological level is thedenaturation of proteins.

Episome: A circular DNA molecule of that can replicate independently ofthe cellular chromosome or integrate and replicate as part of thechromosome.

Gene: DNA fragment that contains sequences necessary to code for anmRNA, and to control the expression of these sequences.

Genome: The total set of genes of an organism enclosed, among theeukaryotes, in chromosomal structures.

Cyclic Probe Reaction: CPT reactions are performed at a constantspecific temperature, which allows hybridization of the chimeric probewith its complementary single-stranded target DNA. Within the resultingtarget-probe duplex, RNase H recognizes the DNA-RNA hybrid andspecifically cleaves the RNA portion of the probe. The cleaved fragmentsare not stable at the reaction temperature and disassociate from thetarget. The target is then free to hybridize with another probemolecule, and the cycle is repeated. The probe fragments accumulate,serving as a basis for the detection of target. Over time, theaccumulation of cleaved probe fragments follows linear kinetics andtherefore the amount of target can be quantified.

Hybridization: A widely used technique that exploits the ability ofcomplementary sequences in single-stranded DNAs or RNAs to pair witheach other to form a double helix. Hybridization can take place betweentwo complimentary DNA sequences, between a single-stranded DNA and acomplementary RNA, or between two RNA sequences. The technique is usedto detect and isolate specific sequences, measure homology, or defineother characteristics of one or both strands.

Infection: Invasion and multiplication of microorganisms in bodytissues, which may be clinically unapparent or result in local cellularinjury due to competitive metabolism, toxins, intracellular replicationor antigen antibody response.

Ligase Chain Reaction: A method of DNA amplification similar to PCR. LCRdiffers from PCR because it amplifies the probe molecule rather thanproducing amplicon through polymerization of nucleotides. Two probes areused per each DNA strand and are ligated together to form a singleprobe. LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme todrive the reaction. Like PCR, LCR requires a thermal cycler to drive thereaction and each cycle results in a doubling of the target nucleic acidmolecule. LCR can have greater specificity than PCR.

Nested PCR: A second PCR that is performed on the product of an earlierPCR using primer, which are internal to the originals. Thissignificantly improves the sensitivity and specificity of the PCR.

Nested primer: A selected primer internal to an amplicon obtained with afirst PCR cycle. The amplification process that uses at least one nestedprimer improves specificity, because the non-specific products of thefirst cycle are not amplified in the second cycle.

Nucleic Acid: Linear polymers of nucleotides, linked by 3′, 5′phosphodiester linkages. In DNA, deoxyribonucleic acid, the sugar groupis deoxyribose and the bases of the nucleotides adenine, guanine,thymine and cytosine. RNA, ribonucleic acid, has ribose as the sugar anduracil replaces thymine. DNA functions as a stable repository of geneticinformation in the form of base sequence. RNA has a similar function insome viruses but more usually serves as an informational intermediate(mRNA), a transporter of amino acids (tRNA), in a structural capacityor, in some newly discovered instances, as an enzyme.

Oligonucleotide/Polynucleotide: Linear sequence of two or morenucleotides joined by phosphodiester bonds. Above a length of about 20nucleotides the term “polynucleotide” is generally used.

Pathogenic agent: A pathogen is a biological agent that can causedisease to its host. A synonym of pathogen is “infectious agent”. Theterm “pathogen” is most often used for agents that disrupt the normalphysiology of a multicellular organism.

Pellet: Sediment, when cells are present, usually includes the cellfraction, or that can be obtained by centrifuging a biological sample.

Polymerase: Enzyme utilized in the amplification of nucleic acids. Theterm includes all of the variants of DNA polymerases.

Primer: Short pre-existing polynucleotide chain to which newdeoxyribonucleotides can be added by DNA polymerase.

PCR: Polymerase Chain Reaction involving two synthetic oligonucleotideprimers, which are complementary to two regions of the target DNA (onefor each strand) to be amplified, are added to the target DNA (that neednot be pure), in the presence of excess deoxynucleotides and Taqpolymerase, a heat stable DNA polymerase. In a series (typically 30) oftemperature cycles, the target DNA is repeatedly denatured (around 90°C.), annealed to the primers (typically at 50-60° C.) and a daughterstrand extended from the primers (72° C.). As the daughter strandsthemselves act as templates for subsequent cycles, DNA fragmentsmatching both primers are amplified exponentially, rather than linearly.

Probe: General term for a fragment of DNA or RNA corresponding to a geneor sequence of interest, that has been labelled either radioactively orwith some other detectable molecule, such as biotin, digoxygenin orfluorescein.

Purification/Decontamination/Sterilization: Refers to a process forremoving contaminants from a sample, where the result is a samplecontaining 60%, preferably 75%, and even more preferably 90% of thematerial toward which the purification procedure is directed.

Restriction Fragment Length Polymorphism (RFLP): A method that allowsgenetic relationship established by comparing the characteristicpolymorphic patterns that are obtained when certain regions of genomicDNA are amplified (typically by PCR) and cut with certain restrictionenzymes. Variations in such patterns are generated by mutations thatcreate or abolish recognition sites for these enzymes

Sample: The term is broadly interpreted and includes any form thatcontains nucleic acids (DNA or RNA) in solution or attached to a solidsubstrate, where the definition of “nucleic acids” includes genomic DNA(for example, when it is attached to a solid substrate, such as in theSouthern Blot or in solution), cDNA, and other forms. Combinations oftwo nucleic-acid sequences through hybridization are formed thanks tothe hydrogen bonds between G and C or A and T bases or analogs of thesebases. These combinations are complementary, and the DNA helixes areanti-parallel. This hybridization combination can be created with onesequence (or helix) in a solution and the other attached to a solidphase (such as, for example, in the FISH [fluorescent in situhybridization] method), or else with both of the sequences in solution.

Single-Strand Conformation Polymorphism (SSCP): SSCP is theelectrophoretic separation of single-stranded nucleic acids based onsubtle differences in sequence (often a single base pair) that resultsin a different secondary structure and a measurable difference inmobility through a gel.

Strand Displacement Amplification (STA): STA is an isothermal, in vitronucleic acid amplification technique based upon the ability of HincII tonick the unmodified strand of a hemiphosphorothioate form of itsrecognition site, and the ability of exonuclease deficient Klenowfragment of DNA Polymerase (exo-klenow) to extend the 3′-end at the nickand displace the downstream DNA strand. Exponential amplificationresults from coupling sense and antisense reactions in which strandsdisplaced from a sense reaction serve as target for an antisensereaction and vice versa.

Target sequence: Nucleic-acid sequence that should be analyzed throughhybridization, amplification, or other methods or combinations ofmethods.

Tm (melting temperature): Temperature at which a specific double-helixDNA population dissociates into single-strand polymers. The formula forcalculating this temperature for polynucleotide fragments is well knownin the art: Tm=81.5+0.41 (% G+C) (Anderson & Young, “Quantitative FilterHybridization,” in Nucleic Acid Hybridization [1985]). Foroligonucleotides with fewer than 40 base pairs, a simplified formula canbe used: Tm=3° C.×(G+C)+2×(A+T).

Tr-DNA/RNA: Transrenal DNA/RNA, or DNA/RNA present in urine after havingbeen passed through the kidney barrier.

Urinary tract: Includes the organs and ducts that participate in theelimination of urine from the body.

Transrenal Nucleic Acids (TrNAs) in Viral Infection

The present invention is based on the discovery that following a viralinfection, the nucleic acids of the virus(es) or of viral origin arecleaved to a relatively short fragments which cross the transrenalbarrier (these nucleic acids are generally termed TrNA, or TrDNA orTrRNA) and can be detected in urine as cell-free low-molecular-weightfragments (whose length is less than 1000 nucleotides) through molecularmethods. As used herein, the term “viral nucleic acid” encompassesnucleic acids of viral origin.

The presence of transrenal nucleic acids (Tr-NA) in urine was detectedpreviously only in hosts who had undergone heterologous tissue or organtransplants, in the case of women pregnant with male fetuses, and in thecase of tumors characterized by specific marker genes. The presence oftransrenal nucleic acids of viral origin in the case of viral infectionsaccording to the present invention is also, and preferably, detected inthe case of non-urinary-tract infections, even in the absence ofhematuria or of pathologies that lead to the rupture, or that alter thenormal integrity, of the renal barrier.

Transrenal nucleic acids (Tr-NA) of viral origin are not associatedwith, and are not derived from, the genome of cells that are lost orreleased in the urinary tract and that are found in urine. Instead, thenucleic acids according to the present invention are filtered by theglomerular-renal filtration mechanism. Thus, the dimensions of thetransrenal nucleic-acid fragments are generally smaller than about 1000base pairs, e.g., smaller than about 500, smaller than about 300,smaller than about 250, or between about 100 and about 200 base pairs,as opposed to other situations in which DNA usually has a high molecularweight and a length in excess of 1000 bases or base pairs.

Therefore, in the present invention, the transrenal nucleic acid (TrNA)of viral origin is generally not found in the urine sediment, but in thesoluble fraction, although traces of TrNA can co-sediment with the cellsduring centrifuging.

Thus, the discovery makes it possible, for the first time, to confirmthe presence of transrenal viral NA directly in urine, and thus isapplicable to the diagnosis and monitoring of any infectious diseasethat has a viral etiology.

Therefore, in embodiments, the invention relates to methods fordiagnosis or monitoring of viral infection by determining the presenceof viral nucleic acids, preferably viral DNA or NA of viral origin, in aurine sample. The methods includes the step of determining the presenceof transrenal viral NA using methods generally used in laboratorypractice such as hybridization, PCR, nested PCR, SSCP, LCR, and SDA.

In certain embodiments, the methods according to the invention includean initial treatment of the urine sample prior to the determination ofthe presence of transrenal viral nucleic acids. In an embodiment, theinvention includes the pretreatment of the urine sample with an agentthat inhibits the degradation of the DNA or RNA. These agents includethe enzymatic inhibitors, such as chelating agents, detergents, ordenaturing agents, DNase or RNase inhibitors, which are preferablyselected from the group consisting of EDTA, guanidine HCl, guanidineisothiocyanate, N-lauryl sarcosine, and sodium dodecyl sulfate.

In another embodiment, the determination of the presence of transrenalviral nucleic acids optionally be preceded by centrifugation orfiltration of the urine sample in order to separate the cellularfraction of the urine from the cell-free low-molecular-weight nucleicacids (DNA/RNA). However, the urine sample may also be utilized withoutfractionation. Centrifugation is preferably performed at a speed between2500 g and 4500 g, and more preferably between 3000 g and 4000 g.Filtration is preferred to carry out through a filter with pore sizebetween 0.1 and 5.0 μm, more preferably with pore size between 0.2 and1.0 μm and even more preferably 0.45 and 0.8 μm. Equivalent methods forseparating the soluble fraction from the cellular fraction may also beused.

Yet in another further embodiment, step b) may optionally be preceded byisolation and/or purification of transrenal nucleic acids. In its turnisolation/purification step may be optionally preceded by filtration orcentrifugation or equivalent technique of urine fractionation.

The isolation and/or purification of the transrenal nucleic acids isachieved through the use of chemical or physical methods that arealready known in the art. It includes one or more purification stepsusing methods selected from among extraction with organic solvents,filtration, precipitation, absorption on solid matrices (e.g., silicaresin, hydroxyapatite or ion exchange), affinity chromatography (e.g.,via sequence specific capture or nucleic acid specific ligands), or elsemolecular exclusion chromatography. However, the purification methodmust be appropriate for the isolation of DNA (single- or double-strand)whose dimensions are smaller than 1000 nucleotide pairs. Even morepreferably, the purification is specific for fragments that are smallerthan 500 nucleotides, and even more preferably, fragments whose lengthare less than 300 or 250 base pairs, or that are between 100 and 200bases or base pairs. The purification preferably takes place on a matrixconsisting but not limited to a silica resin.

In one preferred embodiment, the DNA isolation method is implemented bypretreating the urine sample with a denaturing agent, as describedabove, e.g., urea, guanidine HCl, or guanidine isothiocyanate, at roomtemperature. Guanidine isothiocyanate is preferably utilized. The sampleis then passed through a solid phase, preferably a matrix consisting ofa silica resin that, in the presence of chaotropic salts (guanidineisothiocyanate), binds the nucleic acids. The sample is then collectedor eluted in a buffer, such as Tris-EDTA (Tris 10 mM, EDTA 1 mM), or inwater.

In another preferred embodiment, the characterization and thedetermination of the presence of transrenal viral NA in step b) areperformed through a technique selected from the group consisting of:hybridization of the nucleic acids, a cycling probe reaction (F.Bekkaoui et al., in BioTechniques 20:240-248 [1996]), a polymerase chainreaction (PCR Protocols: A Guide to Methods and Applications, by M.Innis et al.; Elsevier Publications, 1990), a nested polymerase chainreaction, single-strand conformation polymorphism, a ligase chainreaction (LCR) (F. Barany, in PNAS USA, 88:189-93 [1991]), stranddisplacement amplification (SDA) (G. K. Terrance Walker, et al., inNucleic Acid Res, 22:2670-77 [1994], and restriction fragments lengthpolymorphism (RFLP). A technician in the field might also usecombinations of these methods, e.g., PCR-Restriction LengthPolymorphism, in which the nucleic acids are amplified, and then dividedinto aliquots and digested with restriction enzymes, and then separatedvia electrophoresis.

Polymerase chain reaction (PCR) is the preferred method for thedetection and/or quantitative analysis of nucleic acids. Yet morepreferred is the nested PCR method, as defined above, or the semi-nestedPCR method, in which only one of the two primers is internal to theamplicon.

The advantage of the method is linked primarily to the ease ofcollecting the biological samples; to the fact that the transrenalnucleic acids are not infectious; and to the sensitivity of themolecular diagnostic method that can be applied to the nucleic acids,even in the form of fragments.

The diagnostic method is applicable to all viral pathogenic agents,including RNA, DNA, episomal, or integrated viruses. It also applies torecombinant viruses, such as the adenoviruses or lentiviruses utilizedin gene therapy. In particular, the method preferably applies to thefollowing viruses: recombinant and natural HIV-1, HIV-2, variola virus,poliovirus, herpes simplex virus (HSV), Epstein-Barr virus (EBV),hepatitis C virus (HCV), hepatitis B virus (HBV) and adenoviruses (AAV).

The method is preferably applied to the HIV-1 virus, with the selectionof probes or primer oligonucleotides in the GAG, POL, or TAT region forthe detection. Particularly preferred pairs of primers are the onesconsisting of the specific sequences for the HIV GAG region, andpreferably the ones corresponding to the IDN 1-4 sequence, and the onesthat are specific for the HIV-1 POL region, preferably the onescorresponding to the IDN 5-7 region, when the detection is performed viaa polymerase chain reaction (PCR), and, in particular, via nested (GAG)or semi-nested (POL) PCR.

In another of its embodiments, the invention relates to a kit for thedetection and monitoring of transrenal viral NA in urine, including:reagents and/or materials for the separation and/or purification oftransrenal DNA from a urine sample, DNA probes, or pairs of specificoligonucleotides (primers) for at least one viral agent. Reaction tubes,agents for the pretreatment of the sample, enzymes for labeling theprobe, and enzymes for the amplification of the DNA may optionally bepresent.

In a preferred embodiment, the kit includes pairs of oligonucleotideprimers that are specific for recombinant and natural HIV-1, HIV-2,variola virus, poliovirus, herpes simplex virus (HSV), Epstein-Barrvirus (EBV), hepatitis C virus (HCV), hepatitis B virus (HBV),adenoviruses (AAV); or, yet more preferably, primers that are selectedfrom the group consisting of the sequences listed below, and specificreagents for the polymerization chain reaction, preferably in nested orsemi-nested form.

EXAMPLES

The technician in the field may modify all of the methodology describedherein with no change in the basic principal idea.

Example 1 Preparation of the Urine Samples

The method for the preparation of the urine samples and for theextraction of the DNA is described in PCT patent No. WO 98/54364, whichis incorporated by reference in its entirety. All of the steps of thepreparation of the urine samples and of the analysis of the transrenalDNA were performed at room temperature. Briefly, approximately 50-60 mlof urine samples were collected from each patient participating in thestudy. Within 30 minutes after collection, a solution consisting of 0.5MEDTA and 0.5M Tris-HCl, at a pH of 8.5 and at a final concentration of10 mM, was added in order to inhibit the nucleases that might be presentin urine samples. The EDTA has the effect of inhibiting the nucleasesassociated with divalent ions, while the high pH inhibits the acidnucleases.

The stabilized urine samples can be stored, in aliquots of 5 ml, at −80°C. In some instances, the samples were centrifuged for 15 minutes at3500 g, and in this case the extraction and the analysis of the DNA wereperformed on both the supernatant and the sediment.

Example 2 Extraction of the DNA from the Urine Samples

In this study, no commercial kits were used to extract the DNA. In fact,most of the commercial kits utilized for DNA purification are designedto isolate high-molecular-weight DNA, starting from various types ofbiological material.

Bearing in mind that transrenal DNA has a relatively low molecularweight (approximately 150-200 bp), fragments of this size can beisolated from the soluble portion of the urine, even if, in a majorityof cases, the DNA is isolated from non-fractionated urine samples,because a portion of the Tr-DNA could be lost, due to the tendency ofthe Tr-DNA to co-sediment with cells and particulate material during thecentrifugation of the urine sample.

The transrenal DNA was isolated by adding two volumes of 6 M guanidineisothiocyanate to 5 ml of whole urine, or to an equal volume of thefractions previously obtained through centrifugation. The resultingsolution was mixed vigorously.

The soluble low-molecular-weight DNA was captured by Wizard resin(Wizard Resin Suspension, Promega), transferred to a mini-column andwashed thoroughly (through repeated use of large volumes of the washingbuffer supplied with the resin). The Tr-DNA was eluted with water orwith 10 mM Tris-HCl at a pH of 7.5-1 mM EDTA.

Example 3 Design of PCR Primers

The primers for analysis of Tr-DNA based on the use of PCR were selectedfor two different sizes of the target fragment, i.e., one in the rangefrom 60 to 120 bp and the other in the range from 250 to 400 bp. All ofthe primers were also compared against the complete human genomesequence. The primers were designed using the FastPCR software package(biocenter.helsinki.fi/bi/bare-1_html/oligos). The primers for thenested-PCR analysis were selected using the Primer 3 package, which isavailable at the frodo.wi.mit.edu/cg-1-bin/primer3/primer3_www.cgi site,in such a way that the melting temperature of the internal, nestedprimers was not lower than that of the external primers.

The HIV primers were selected for recognition of all 9 of the Type M HIVsubtypes. In accordance with the recently revised nomenclature (i.e.,the 1999 Nomenclature Proposal:[hiv.lan1.gov/content/hiv-db/HTML/reviews/nomenclature/Nomen]), theHIV-1 M group subtypes are represented by phylogenetically associatedgroups of HIV-1 sequences. They are designated as A1, A2, B, C, D, F1,F2, G, H, J, and K. The M group contains viruses that representapproximately 95% of all of the cases of HIV in Europe. Complete genomicsequences of HIV-1 M group subtypes were obtained from the database atthe Los Alamos National Laboratory (New Mexico, hiv.lanl.gov). Themultiple alignments and the creation of consensus sequences of thevarious subtypes were performed through the use of ClustalX algorithmincluded in the BioEdit package (mbio.ncsu.edu/BioEdit/bioedit.html).

The following primers were selected:

GAG Primers for Nested PCR.

External: GAG 468-F (SEQ ID NO: 1): TGGGTAAAAGTAATAGAGGAGAAGGC; GAG602-R (SEQ ID NO: 2): AACATTTGCATGGCTGCTT.Product: 134 bp.

Internal: GAG 518-F (SEQ ID NO: 3): CAGCATTATCAGAAGGAGCCACC; GAG 596-R(SEQ ID NO: 4): TGCATGGCTGCTTGATGTCC.Product: 79 bp.POL primers for semi-nested-PCR, short amplicon

External primers: POL 4368-F (SEQ ID NO: 5): GGRGAAGCCWTGCATGGAC; POL4468-R (SEQ ID NO: 6): GCTACATGRACTGCTACCAG.Product: 102 bp.

Internal primers: POL 4368-F (SEQ ID NO: 5): GGRGAAGCCWTGCATGGAC; POL4427-Rn (SEQ ID NO: 7): TGTRCAATCTARTTGCCATATYCCTGG.Product: 60 bp.Pol Primers for Semi-Nested-PCR, Long Amplicon

External primers: POL 4368-F (SEQ ID NO: 5): GGRGAAGCCWTGCATGGAC POL4678-R (SEQ ID NO: 8): ACTCCYTGRCTTTGGGGATTGProduct: 311 bp.

Internal primers (nested): POL 4368-F (SEQ ID NO: 5):GGRGAAGCCWTGCATGGAC POL 4427-Rn (SEQ ID NO: 7):TGTRCAATCTARTTGCCATATYCCTGG.Product: 60 bp.TAT primers for semi-nested PCR (long amplicon)

External primers: TAT 5955-F (SEQ ID NO: 9): GCTTAGGCATYTCCTATGGCAG TAT6462-R (SEQ ID NO: 10): TGGGGTCTGTKGGTACACAGG.Product: 569 bp.

Internal primers: TAT 6330-Fn (SEQ ID NO: 11):CWGTHTAYTATGGRGTACCTGTGTGG TAT 6462-R (SEQ ID NO: 10):TGGGGTCTGTKGGTACACAGG.Product: 132 bp.

Example 4 Diagnosis of HIV-1 Infection Based on TrDNA

The DNA was isolated from the urine samples of 10 patients infected withHIV, as described in the preceding examples. These patients wereclinically diagnosed as being infected with HIV through the use ofstandard clinical molecular tests to detect the presence of antibodiesthat are specific for the HIV virus.

FIG. 1 shows the electrophoresis of the HIV-1 DNA fragments, asamplified via nested PCR, with the above-indicated two pairs ofGAG-specific primers, in accordance with the method described in theinvention. A band whose dimensions correspond to the ones that wereexpected as the result of the nested PCR amplification—as performed withthe use of the 468/602 pair of primers in the first amplification,followed by the 518/596 pair (79 bp)—was observed in the urine of 8 ofthe 10 patients and in the positive controls, but not in the negativecontrols.

Table 1 indicates the viral load of each of the patients who wereanalyzed, from which it can be inferred that in the case of the samples(numbers 2 and 3) that were negative upon amplification, the viral loadwas lower than 50 copies of the virus per ml of plasma. TABLE 1 Viralload of the HIV-infected patients. Patient No. Infection HIV load 1 HIV120,000 2 HIV <50 3 HIV <50 4 HIV 867 5 HIV 88,000 6 HIV 8,370 7 HIV32,046 8 HIV + HCV 500,000 9 HIV 15,822 10 HIV + HCV <50

The viral load was determined by measuring the quantity of theHIV-specific RNA via RT-PCR in the plasma.

The apparent sensitivity of the nested PCR was evaluated by amplifying,with the same pair of primers, an 8E5 LAV cell line genomic DNA carryinga single integrated copy of the HIV genome. The sensitivity wasdetermined to be 5 genome equivalents. Sequence list GAG 468-F (seq. No.1): TGGGTAAAAGTAATAGAGGAGAAGGC; GAG 602-R (seq. No. 2):AACATTTGCATGGCTGCTT; GAG 518-F (seq. No. 3): CAGCATTATCAGAAGGAGCCACC;GAG 596-R (seq. No. 4): TGCATGGCTGCTTGATGTCC. POL 4368-F (seq. No. 5):GGR*GAAGCCW*TGCATGGAC; POL 4468-R (seq. No. 6): GCTACATGR*ACTGCTACCAG;POL 4427-Rn (seq. No. 7): TGTRCAATCTARTTGCCATATY*CCTGG POL 4678-R (seq.No. 8): ACTCCY*TGR*CTTTGGGGATTG TAT 5955-F (seq. No. 9):GCTTAGGCATY*TCCTATGGCAG TAT 6462-R (seq. No. 10): TGGGGTCTGTK*GGTACACAGGTAT 6330-Fn (seq. No. 11): CW*GTHTAY*TATGGRGTACCTGTGTGG Nucleotidesindicated by an asterisk (*) indicate degenerate nucleotides known tothose skilled in the art.

Equivalents

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the precise form ofthe disclosed invention or to the scope of the appended claims thatfollow. In particular, it is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims. Various alterations and modifications of theinvention are believed to be a matter of routine for a person ofordinary skill in the art with knowledge of the embodiments describedherein. Other aspects, advantages, and modifications considered to bewithin the scope of the following claims.

1. A method for diagnosis of a viral infection in a subject, such methodcomprising detecting the presence of viral transrenal nucleic acids in aurine sample from said subject, thereby diagnosing a viral infection insaid subject.
 2. The method according to claim 1, in which said nucleicacids are deoxyribonucleic acid (DNA).
 3. The method according to claim1, further comprising the step of quantitating said viral transrenalnucleic acids.
 4. The method according to claim 3, wherein saidquantitating is performed by a means selected from the group consistingof pairing with molecular probes that are specific for those pathogenicagents, hybridization, PCR, nested PCR, SSCP, LCR, and SDA.
 5. Themethod according to claim 1, further comprising the step of separatingthe urine sample into a cell-free fraction containing said transrenalnucleic acids.
 6. The method according to claim 5, in which saidseparation is selected from the group consisting of filtration andcentrifugation of said urine sample.
 7. The method according to claim 5,in which the length of said viral transrenal nucleic acids is less thanthat about 1000 nucleotides.
 8. The method according to claim 5, inwhich said viral transrenal nucleic acids comprise DNA fragments, andthe length of said fragments is between about 100 and about 200 bp. 9.The method according to claim 1, further comprising the step ofisolation or purification of said nucleic acids.
 10. The methodaccording to claim 9, wherein said purification is performed throughchemical or physical methods.
 11. The method according to claim 9, inwhich said isolation or purification is performed using a methodselected from the group consisting of extraction with organic solvents,filtration, precipitation, absorption on solid matrices having anaffinity for the nucleic acids, and chromatography.
 12. The methodaccording to claim 11, in which said solid matrices consist ofsilica-based resins, ion-exchange resins, or hydroxyapatite.
 13. Themethod according to claim 12, in which said solid matrix is asilica-based resin, and said isolation or purification is performed inthe presence of a chaotropic agent.
 14. The method according to claim 1,including a pretreatment of the urine sample with one or more agentsthat inhibit the degradation of the nucleic acids.
 15. The methodaccording to claim 14, in which said agents are selected from the groupconsisting of ion-chelating agents, denaturing agents, and ionicdetergents.
 16. The method according to claim 15, in which saidion-chelating agents are EDTA; said denaturing agents are guanidine HClor guanidine isothiocyanate; and said ionic detergents are N-laurylsarcosine or sodium dodecyl sulfate.
 17. The method according to claim1, wherein said detecting the presence of said viral nucleic acids isperformed through a technique selected from the group consisting ofhybridization of the nucleic acids, a cycling probe reaction, apolymerase chain reaction, a nested polymerase chain reaction,single-strand conformation polymorphism, a ligase chain reaction, stranddisplacement amplification, and restriction fragments lengthpolymorphism.
 18. The method according to claim 17, in which said PCR isnested or semi-nested PCR.
 19. The method according to claim 1, in whichsaid viral nucleic acid is derived from an RNA or a DNA virus.
 20. Themethod according to claim 19, in which said virus is integrated orepisomal.
 21. The method according to claim 19, wherein said virus isselected from the group consisting of recombinant and natural HIV-1,HIV-2, variola virus, poliovirus, herpes simplex virus (HSV),Epstein-Barr virus (EBV), hepatitis C virus (HCV), hepatitis B virus(HBV), and adenovirus (AAV).
 22. The method according to claim 19,wherein said virus is selected from the group consisting of EBV andHIV-1.
 23. The method according to claim 19, wherein said virus isHIV-1.
 24. The method according to claim 9, in which said isolated orpurified nucleic acid is used for the detection of said viral transrenalnucleic acid.
 25. The method according to claim 1, wherein said methodis performed in vitro and said viral nucleic acid is an HIV nucleicacid.
 26. The method according to claim 25, wherein said detection ofsaid viral nucleic acid is performed through a polymerase chain reaction(PCR).
 27. The method according to claim 26, in which said PCR is nestedor semi-nested PCR.
 28. The method according to claim 24, in which saidpolymerase chain reaction is performed using one or more primers thatare specific for the HIV-1 GAG or POL gene.
 29. The method according toclaim 27, in which the one or more primers are selected from the groupconsisting of SEQ ID NOs: 1 to
 11. 30. A method for monitoring a viralinfection in a subject, such method comprising: a) quantitating theamount of a viral transrenal nucleic acid in a first urine sample fromsaid subject; b) quantitating the amount of said viral transrenalnucleic acid in a second urine sample from said subject; and c)comparing the amount of said viral transrenal nucleic acid in said firstand said second urine sample from said subject, thereby monitoring saidviral infection in said subject.
 31. The method according to claim 30,further comprising the step of administering to said subject a compoundthat reduces or inhibits said viral infection.
 32. The method accordingto claim 31, wherein said viral infection is HIV infection and saidcompound is an anti-viral agent.
 33. The method according to claim 30,wherein said quantitating is performed by a means selected from thegroup consisting of pairing with molecular probes that are specific forthose pathogenic agents, hybridization, PCR, nested PCR, SSCP, LCR, andSDA.
 34. The method according to claim 30, further comprising the stepof separating said urine samples into a cell-free fraction containingsaid transrenal nucleic acids.
 35. The method according to claim 34, inwhich said separation includes centrifugation of said urine sample. 36.The method according to claim 30, in which said viral transrenal nucleicacids comprise DNA fragments, and the length of said fragments isbetween about 100 and about 200 bp.
 37. The method according to claim30, wherein said subject is at risk of developing a recurring viralinfection.
 38. A kit for the determination of the presence of a viralnucleic acid in a urine sample, comprising means for isolation orpurification of said nucleic acid from said urine sample, and means fordetermination of the presence of said nucleic acid by hybridization witha virus-specific probe.
 39. The kit according to claim 38, in which saidvirus-specific probe comprises a primer for the polymerization chainreaction.
 40. The kit according to claim 39, in which said primer isspecific for the HIV-1 GAG or POL gene.
 41. The kit according to claim38, in which said probe is selected from the group consisting of SEQ IDNO: 1 to 11.